Influenza virus is a seasonal pathogen with the potential to cause pandemic infection;it results in respiratory disease ranging from subclinical symptoms to life-threatening primary viral pneumonia. Despite the availability of a yearly vaccine, it is estimated that an average of 360,000 deaths/year can be attributed to influenza virus infection in the US alone, with severe illness occurring in approximately 3-5 million people worldwide annually. Through the use of small regulatory RNAs, we seek to further the current paradigm of influenza virus infection while developing a safe, effective, and cost-efficient vaccine strain. Influenza A virus is not only a seasonal pathogen, causing yearly epidemics, but also a potential pandemic causative agent. The development of reverse genetics technology for influenza virus has led both to the preliminary characterization of its pathogenicity, as well as to the development of novel vaccine strains. By combining reverse genetics with the newly evolving field of microRNA (miRNA) biology, we seek to further understand influenza virus infection while developing a safe, effective, and easily produced vaccine strain. Through the incorporation of miRNA response elements (MREs), corresponding to ubiquitously expressed mammalian miRNAs, into influenza A virus, we can achieve vaccine quality attenuation. Our first objective will be to characterize this phenomenon, by fully understanding the host response to these attenuated viruses, and by elucidating the viral response to these attenuating mutations. We will also improve upon this technology by identifying the most effective influenza viral transcript to target, with the ultimate goal being influenza virus vaccine design. Our second objective will be to use this new technology to investigate influenza virus tropism and cell-type contribution to viral pathogenicity. Through the use of miRNA-mediated attenuation technology, we will elucidate the role of hematopoietic-specific miRNAs in determining influenza virus tropism, and further our understanding of the contribution made by epithelial subtype populations to influenza virus pathogenicity. As such, this proposal has the potential to offer a significant contribution to human health as the strategy described here could be employed to develop effective and safe vaccines to any desired pathogen.